Difference between revisions of "Part:BBa K2350009"
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Figure 1.  
 
Figure 1.  
  
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C1~C20 represents the pPIGBACK-CrtZ transformant clone1 to clone20, and M represnets 1 kb marker.
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Figure 1 is pPIGBACK-CrtZ transformants electrophoresis result. C1~C20 represents the pPIGBACK-CrtZ transformant clone 1 to clone 20, and M represnets 1 kb marker.
  
  

Revision as of 12:10, 25 October 2017


Pigment double-crossover homologous gene recombination bacbone (pPIGBACK)

The vector, pPIGBACK, is used to transform into S. elongatus PCC 7942 with the inserted pigment gene in our project. pPIGBACK contains 5’- and 3’-ends of the neutral site II (NSII), replication origin of pBR322, ampicillin resistance gene, and double terminator BBa_B0015. The strategy we chose to construct the vector is to fuse B0015 and AmpR together first. Secondly, we fused 5’- and 3’-ends of the neutral site II (NSII) with PBR322 replication origin (ORI) together. At last, we ligated two parts together. The aim of constructing this vector is to finish double-crossover homologous gene recombination in S. elongatus PCC 7942. To insert BBa_J04450 with EcoR1 and Pst1 cutting sites, we use site-directed mutagenesis to remove all Pst1 cutting sites in pPIGBACK.


Transformation efficiency of Ppigback-CrtZ is 11.4 transformants per μg DNA, and correctness is 52% (10/19), which is quite efficient because the successful rate of gene double-crossingover homologous recombination is low. See figure 1.



Figure 1.

Figure 1 is pPIGBACK-CrtZ transformants electrophoresis result. C1~C20 represents the pPIGBACK-CrtZ transformant clone 1 to clone 20, and M represnets 1 kb marker.


CrtZ Parts.jpg





Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found at 1
    Illegal suffix found at 3880
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 1
    Illegal SpeI site found at 3881
    Illegal PstI site found at 3895
    Illegal NotI site found at 7
    Illegal NotI site found at 1321
    Illegal NotI site found at 3888
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 1
    Illegal BglII site found at 1883
    Illegal BamHI site found at 2180
    Illegal XhoI site found at 1118
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found at 1
    Illegal suffix found at 3881
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found at 1
    Plasmid lacks a suffix.
    Illegal XbaI site found at 16
    Illegal SpeI site found at 3881
    Illegal PstI site found at 3895
    Illegal NgoMIV site found at 2101
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI site found at 972
    Illegal BsaI site found at 1783